This disclosure relates generally to tests for determining the presence of certain microorganisms and specifically to a test for detecting Neisseria bacteria via an immobilized antibody-enzyme complex, which enzyme is clinically unique to Neisseria bacteria.
The importance of quickly and accurately detecting the presence of Neisseria bacteria, especially Neisseria gonorrhoeae, is well recognized. Present tests for determining the presence of such organisms as N. gonorrhoeae include the preparation of bacterial cultures or the use of serological methods. Such tests, however, have known limitations. See, e.g., the publication, "International Symposium On Gonorrhea," edited by B. B. Diena, which is a collection of papers presented at the October, 1973 International Symposium on Gonorrhea sponsored by the Health Protection Branch, Health and Welfare, Ottawa, Canada, especially at p. 34, et seq.
A relatively simple and quick test for the presence of Neisseria in liquid samples is disclosed in related application Ser. No. 837,366, cited above. Such test is based upon the discovery of an enzyme in Neisseria bacteria which, at the time, appeared to be specific thereto. The structure of the enzyme is not fully understood and no identification thereof in the literature is known. However, the enzyme has the capability of oxidizing 1,2-propanediol and reducing nicotinamide-adenine-dinucleotide (NAD). Because of these two characteristics, the enzyme was named 1,2-propanediol dehydrogenase, which name will be used throughout this specification.
That a biochemical reaction can take place between an antigen and its homologous antibody, giving rise to an antibody-antigen complex, is well recognized. It also is well known that enzymes typically are antigenic and that, as a consequence, antibody-enzyme complexes are readily prepared. See, e.g., M. R. J. Salton, Editor, "Immunochemistry of Enzymes and Their Antibodies," John Wiley & Sons, New York, 1977; B. Cinader, Editor, "Antibodies to Biologically Active Molecules," Vol. 1 of the Proceedings of the 2nd Meeting of the Federation of European Biochemical Societies, Vienna, 21-24 April 1965, Symposium Publications Division, Pergamon Press, Oxford, 1967; B. Cinader, Consulting Editor, "Antibody to Enzymes--A Three-Component System," Ann. N.Y. Acad. Sci., 103 (Art. 2), 493-1154 (1963); and C. A. Williams and M. W. Chase, Editors, "Methods In Immunology and Immunochemistry," Vol. IV, Academic Press, New York, 1977, pp. 312-375. In the case of an enzyme, however, formation of the antibody-enzyme complex frequently results in the inhibition of enzymatic activity. Such inactivation, though, can be employed as the basis of an assay for a particular enzyme. That is, the presence of the enzyme in a sample can be detected by adding to the sample antibody specific for the enzyme and monitoring the resulting mixture for a decrease in enzymatic activity.
Inhibition of the enzymatic activity of 1,2-propanediol dehydrogenase, the above-described enzyme which is clinically unique to Neisseria bacteria, is known to occur in the presence of antibodies specific for the enzyme. Indeed, this inhibition is an important factor in three of the related applications cited hereinbefore.
Application Ser. No. 837,364 discloses a method of using antibodies directed against an enzyme present in Neisseria, i.e., 1,2-propanediol dehydrogenase, to inhibit enzyme activity in a sample, thereby inferring antibody specificity on the assay for Neisseria bacteria which is in fact a test for the presence of such enzyme. The percent inhibition of the enzyme is shown to be directly proportional to the incubation time and the amount of antibody present per unit of enzyme activity. For example, at a globulin:enzyme ratio of 1.47 (mg. total globulin in the antibody preparation per International Unit of enzyme), the percent inhibition of enzymatic activity after 0.5, 1.0, and 2.0 hours is 15, 23, and 38, respectively. In other examples, the percent inhibition at globulin:enzyme ratios of 0.75 and 0.075 varied from 76 to 91 and from 23.5 to 29, respectively, with the incubation times ranging from 24 to 141 minutes and from 30 to 144 minutes, respectively.
Application Ser. No. 837,363 discloses a modification of the method described in the above application, Ser. No. 837,364, wherein ammonium sulfate precipitation of the antibody-enzyme complex serves to concentrate the complex, thereby removing interfering materials and improving the speed and precision of the method. In a preferred embodiment, the inhibitory effect of the antiserum is explicitly exhibited through a comparative test wherein one sample is contacted with antiserum and a second sample serves as a control. Clearly, the modification requires inhibition of enzymatic activity in the presence of antibody (antiserum) specific for the enzyme.
Finally, application Ser. No. 837,362 discloses two closely-related assay methods for detecting the presence of Neisseria bacteria in a sample. Both methods utilize radiolabelled antibody specific for the enzyme 1,2-propanediol dehydrogenase which is released upon lysis of the Neisseria bacteria. While neither enzymatic activity nor the inhibition thereof are required for the assay, the specification describes the use of such inhibition of enzymatic activity to titer the antiserum. Thus, undiluted antiserum resulted in a 96% inhibition of enzymatic activity. Even with dilutions of up to 1:16, the inhibition of enzymatic activity was 70% or higher.